Compositions and methods for preventing and treating cancer via modulating ube1l, isg15 and/or ubp43

ABSTRACT

Compositions and methods of using compositions that induce UBE1L or a ubiquitin-like protein ISG15 or inhibit a deconjugase UBP43 to degrade oncogenic proteins and enhance apoptosis of cancer (neoplastic) or pre-cancerous (pre-neoplastic) cells are provided. Methods for the prevention or treatment of cancer via administration of these compositions are also provided.

INTRODUCTION

This work was supported in part by National Institutes of Health GrantRO-1-CA62275 and National Institutes of Health Grant RO-1-CA87546 andthe U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

UBE1L, the ubiquitin-like protein ISG15 and the deconjugase UBP43 havenow been identified as direct pharmaceutical targets that overcomeoncogenic effects of oncogenic proteins such as PML/RARα, triggeringdegradation of the oncogenic proteins and signaling apoptosis-in cancercells expressing the oncogenic proteins. The present invention relatesto new compositions as well as methods of designing compositionstargeted to UBE1L and/or ubiquitin-like proteins such as ISG15 or thedeconjugase UBP43 which are useful in enhancing the pro-apoptotic anddegradative pathway that opposes effects of oncogenic proteins such asPML/RARα. The present invention also relates to methods for preventingand treating cancer which involve inducing UBE1L and/or ubiquitin-likeproteins such as ISG15 or, alternatively, inhibiting the deconjugaseUBP43 to increase programmed cell death (apoptosis) in neoplastic andpre-neoplastic cells.

BACKGROUND OF THE INVENTION

Acute promyelocytic leukemia (APL) (FAB M3) cases express the oncogenicproduct of the t(15;17) chromosomal rearrangement, promyelocyticleukemia (PML)/retinoic acid receptorα (RARα) (Nason-Burchenal et al.(1996) in Molecular Biology of Cancer, ed. Bertino, J. R. (Academic SanDiego), 1st Ed., pp 1547-1560; Nason-Burchenal, K. and Dmitrovsky, E.(1999) in Retinoids: The Biochemical and Molecular Basis of Vitamin Aand Retinoid Action, eds. Nau, H. & Blaneer, W. (Springer, Berlin), pp.301-322). All-trans-retinoic acid (RA) treatment causes completeremissions in these APL cases through induction of leukemic celldifferentiation (Nason-Burchenal et al. (1996) in Molecular Biology ofCancer, ed. Bertino, J. R. (Academic San Diego), 1st Ed., pp 1547-1560;Nason-Burchenal, K. and Dmitrovsky, E. (1999) in Retinoids: TheBiochemical and Molecular Basis of Vitamin A and Retinoid Action, eds.Nau, H. & Blaneer, W. (Springer, Berlin), pp. 301-322). A hallmark of RAresponse in APL is PML/RARα degradation that reverses PML/RARα oncogeniceffects (Kakizuka et al. Cell 1991 68: 663-674; de Th6 et al. Cell 199168: 675-684; Yoshida et al. Cancer Res. 1996 56: 2945-2948; Raelson etal. Blood et al. 1996 88: 2826-2832; Nervi et al. Blood 1998 92:2244-2251; Zhu et al. Proc. Natl. Acad. Sci USA 1999 96: 14807-14812).Proteasomal inhibitors prevent PML/RARα proteolysis, despite RAtreatment, which is indicative of a proteasome-dependent pathway in thisdegradation (Yoshida et al. Cancer Res. 1996 56: 2945-2948; Raelson etal. Blood et al. 1996 88: 2826-2832; Nervi et al. Blood 1998 92:2244-2251; Zhu et al. Proc. Natl. Acad. Sci USA 1999 96: 14807-14812).PML/RARα expression results in dominant-negative transcriptionalrepression (Kakizuka et al. Cell 1991 68: 663-674; de The et al. Cell1991 68: 675-684). This repression is antagonized by pharmacological RAdosages that overcome inhibitory effects on transcription of theN-Cor/SMRT corepressor complex that has histone deacetylase activity(Lin et al. Nature (London) 1998 391: 811-814; Grignani et al. Nature(London) 1998 391: 815 818). RA treatment recruits a coactivator complexthat stimulates transcription, resulting in activation of target genes(Lin et al. Nature (London) 1998 391: 811-814; Grignani et al. Nature(London) 1998 391: 815-818).

To understand the molecular basis of RA response in APL, identificationof RA target genes is required.

GOS2 is a putative RA target gene identified by microarray analysis ofAPL cells (Tamayo et al. Proc. Natl. Acad. Sci. USA 1999 96: 2907-2912).The precise function of GOS2 is not yet known, but it was firstidentified as regulated during the cell cycle (Russell, L. and Forsdyke,D. R. DNA Cell Biol. 1991 10: 581-591), suggesting a role in cell cyclecontrol.

Another candidate retinoid target gene is the CCAAT/enhancer bindingprotein ε (C/EBP ε) that contributes to retinoid transcriptional effectsin APL (Park et al. J. Clin. Invest. 1999 103: 1399-1408). However, thisspecies has not been linked to the degradation of PML/RARα.

Recent microarray analysis of RA-treated NB4 APL cells reported theprominent induction of UBE1L (ubiquitin-activating enzymeE1-like)(Tamayo et al. Proc. Natl. Acad. Sci. USA 1999 96: 2907-2912).The proteasome-dependent degradation of PML/RARα has also been proposedas a mechanism by which RA overcomes PML/RARα oncogenic effects (Yoshidaet al. Cancer Res. 1996 56: 2945-2948; Raelson et al. Blood et al. 199688: 2826-2832; Nervi et al. Blood 1998 92: 2244-2251; Zhu et al. Proc.Natl. Acad. Sci USA 1999 96: 14807-14812). UBE1L could account for theproteasome dependent degradation of PML/RARα and perhaps other oncogenicproteins.

Hammerhead ribozymes that target PML/RARα have been used to show howPML/RARα degradation signals apoptosis but not differentiation intransfected APL cells that are either RA-sensitive or RA-resistant(Nason-Burchanel et al. Blood 1998 92: 1758-1767; Nason-Burchanel et al.Oncogene 1998 17: 1759-1768).

It has now been found that UBE1L is a retinoid target gene in APL thatantagonizes PML/RARα oncogenic effects by triggering PML/RARαdegradation. The consequence of this action is the promotion ofapoptosis resulting in anti-oncogenic effects of UBE1L in APL andpotentially in other neoplastic or pre-neoplastic cell contexts.Accordingly, compositions which target UBE1L or other proteins relatedthereto, such as the ubiquitin-like protein ISG15 or the deconjugaseUBP43, are expected to be useful in preventing and treating cancer.

SUMMARY OF THE INVENTION

An object of the present invention is to provide compositions forprevention and treatment of cancer comprising an agent which inducesUBE1L and/or a ubiquitin-like protein such as ISG15 or an agent whichinhibits the deconjugase UBP43.

Another object of the present invention is to provide a method foridentifying agents as potential therapeutics against cancer whichcomprises determining the ability of the agent to induce UBE1L and/orubiquitin proteins such as ISG15 or determining the ability of the agentto inhibit the deconjugase UBP43.

Another object of the present invention is to provide a method forenhancing pro-apoptotic and degradative pathways of neoplastic(cancerous) cells or pre-neoplastic (pre-cancerous) cells with an agentwhich induces UBE1L and/or ubiquitin-like proteins such as ISG15 or anagent which inhibits the deconjugase UBP43.

Another object of the present invention is to provide a method forpreventing or treating cancer in a patient which comprises administeringto a patient an agent which induces UBE1L and/or ubiquitin-like proteinssuch as ISG15 or an agent which inhibits UBP43.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anticancer agents, methods fordesigning new anticancer agents and methods of using agents that induceactivity and/or expression of ubiquitin-activating enzyme E1-likeprotein (UBE1L) and/or ubiquitin-like proteins such as ISG15.Alternatively, agents of the present invention may inhibit the activityand/or expression of the deconjugase UBP43. Agents of the presentinvention are useful in preventing or treating cancerous or neoplasticas well as pre-cancerous or pre-neoplastic cells. UBE1L is widelyexpressed in diverse human tissues and tumor cell lines. UBE1L acts asthe activating enzyme for the ubiquitin-like protein ISG15. Asdemonstrated herein, UBE1L is a RA inducible gene target in acutepromyelocytic leukemia and U937 and THP1 cells, implicating a broadbiological role for UBE1L. Expression of ISG15 as well as thedeconjugase UBP43 have also been found to be induced by RA and appear tobe regulated in a coordinated fashion with UBE1L. As shown herein, thisinduction occurs only in RA sensitive cells, and not in RA insensitivecells. Further, coordinate regulation and physical association of UBE1Land ISG15 induces degradation of oncogenic proteins including, but notlimited to, PML/RARα, cyclin D1, and PML, in RA sensitive cells. Inaddition, the induction of degradation of these oncogenic proteins byUBE1L and ISG15 appears to preferentially trigger apoptosis. PML/RARαdegradation is inhibited by UBP43 transfection. Accordingly, agentswhich induce activity and/or expression of UBE1L or ubiquitin-likeproteins such as ISG15 and agents which inhibit activity and/orexpression of the deconjugase UBP43 are expected to be useful intreatment of neoplasia and pre-neoplasia, particularly RA sensitivecancers and cancers expressing oncogenic proteins such PML/RARα.

All-trans-retinoic acid (RA) treatment induces remissions in acutepromyelocytic leukemia (APL) cases expressing the t(15;17) gene product,promyelocytic leukemia (PML)/retinoic acid receptors (RARα). Microarrayanalyses has revealed induction of UBE1L (ubiquitin-activating enzymeE1-like) after RA treatment of NB4 APL cells. The kinetics of thisinduction was studied in RA-sensitive NB4-S1 APL cells using a reversetranscription-PCR assay. UBE1L mRNA induction occurred by 3 hours after10 μM RA treatment. These results were independently confirmed byNorthern analysis and after 1 μM RA treatment by reversetranscription-PCR assay. In contrast, UBE1L expression was not inducedduring the same time period, despite 10 μM RA treatment of theRA-resistant NB4-R1 cell line. This is indicative of UBE1L being adirect retinoid target.

Further, the direct relationship between UBE1L induction and effectiveretinoid treatment of APL cells was demonstrated by examination of UBE1Limmunoblot expression. For these experiments, immunogenic peptidesdescribed in detail in Example 5 were used to generate independentpolyclonal antisera recognizing the amino or carboxyl termini of UBE1Lprotein, respectively. Chinese Hamster Ovary (CHO) cells that did notbasally express UBE1L mRNA were transfected with a full-length UBE1LcDNA or an insertless vector. CHO cells transfected with UBE1L expressedUBE1L protein. In contrast, cells transfected with an insertless vectordid not express this 112-kDa species. The UBE1L immunoblot expressionprofiles were also compared in RA-sensitive versus RA-resistant NB4cells. UBE1L protein was basally expressed at low levels in both celllines. However, protein expression was induced only after RA (1 μM)treatment of NB4-S1 APL cells.

A hallmark of RA response in APL is PML/RARα degradation (Yoshida et al.Cancer Res. 1996 56: 2945-2948; Raelson et al. Blood et al. 1996 88:2826-2832; Nervi et al. Blood 1998 92: 2244-2251; Zhu et al. Proc. Natl.Acad. Sci USA 1999 96: 14807-14812). RA treatment has been reported torepress PML/RARα expression in NB4-S1, but not in RA-resistant NB4-R1cells (Nason-Burchenal Differentiation 1997 61: 321-331). To examine therelationship in APL cells between UBE1L and PML/RARα expression,immunoblot expression profiles for these species were examined beforeand after 24 hour RA (1 μM) treatment of NB4-S1 cells. An inverserelationship was evident between UBE1L and PML/RARα expression bothbefore and after RA treatment thus indicating a direct role for PML/RARαin regulating UBE1L expression.

A 1.3-kb fragment of the UBE1L promoter was then demonstrated to becapable of mediating transcriptional response to RA in a retinoidreceptor-selective manner. PML/RARα, a repressor of RA target genes,abolished this UBE1L promoter activity. To examine the potential forPML/RARα to affect UBE1L, 1.3 kilobases (kb) of the UBE1L promoterupstream of the ATG translation start site was cloned into aluciferase-containing reporter plasmid. This reporter plasmid wastransfected into CHO cells in the presence and absence of RA treatment.This fragment of the UBE1L promoter was capable of mediatingtranscriptional response to RA in a retinoid receptor-selective manner.

The relationship between PML/RARα and activity of this UBE1L reporterplasmid was examined when PML/RARα was cotransfected with this reporterplasmid. Cotransfection of PML/RARα with RARα led to a marked repressionof UBE1L reporter activity before and after RA (1 μM) treatment. Thisinhibition depended on the dosage of transfected PML/RARα. In eachexperiment a cotransfected β-galactosidase reporter plasmid was used tocontrol for transfection efficiencies. No appreciable effect of PML/RARαon the transcriptional activity of the β-galactosidase reporter plasmidwas observed. Thus, PML/RARα repressed activity of this UBE1L reporterplasmid.

UBE1L has homology to E1. However, E1 mRNA was not induced after RA (1μM) treatment of NB4-S1 cells, thus indicating different effects of RAon expression of UBE1L and E1.

A hallmark of RA response in APL is PML/RARα degradation. Accordingly,the ability of UBE1L as well as E1 to trigger PML/RARα degradation wasnext examined. Cotransfection assays were performed using cells that donot express PML/RARα. In these experiments CHO cells that did notexpress UBE1L and BEAS-2B cells that expressed low levels of UBE1L, butcould be readily transfected with RARs or PML/RARα, were used.Degradation of transfected PML/RARα was triggered by UBE1L in adose-dependent manner after transfection of CHO cells or BEAS-2B cells.This degradation of PML/RARα occurred in the absence of RA treatment.The PML domain of PML/RARα appears to be more sensitive to degradationby UBE1L than the RARα domain. Transfection of a truncated UBE1L(pSG5-UBE1L-T) did not cause PML/RARα degradation. To establish thatPML/RARα degradation was a distinct UBE1L function, E1 was alsotransfected into BEAS-2B cells with PML/RARα. E1 did not cause PML/RARαdegradation. Thus, transfection of UBE1L, but not E1, led to PML/RARαdegradation even without RA treatment.

The effects of engineered overexpression of UBE1L in APL cells on growthor differentiation state of these cells was then examined. Tooverexpress UBE1L in APL cells, retroviral vectors (Ory et al. Proc.Natl. Acad. Sci. USA 1996 93: 11400-11406) were constructed to expressUBE1L or no insert. Coexpressed GFP was used to enrich forretroviral-expressing cells after FACS sorting. HeLa cells, which do notexpress PML/RARα and basally express UBE1L at low levels, were used as acontrol for these experiments because retroviral transduction conditionswere previously optimized in these cells. UBE1L overexpression wasengineered independently in NB4-S1 and HeLa cells using the describedretroviral transduction method. As a control, an insertless controlvector was independently introduced into these cell lines as confirmedby immunoblot analysis. A striking difference in biological effects wasobserved after transduction of UBE1L into NB4-S1 versus HeLa cells.UBE1L overexpression in NB4-S1 cells resulted in the rapid induction ofapoptosis as measured by the Hoechst staining of transduced cells. Threeindependent fields were examined for the insertless control NB4-S1transfectants and 5.1% of these cells were apoptotic. Analysis ofUBE1L-transduced NB4-S1 cells revealed a high proportion (39.7%) ofapoptotic cells. These transductants did not exhibit morphologicalevidence of leukemic cell maturation. This lack of induceddifferentiation was confirmed by the absence of NET-positive cells (seeTable 1).

Table 1: NBT Maturation Assays Performed on NB4-S1 APL Cells After 5Days Treatment with RA (1 μM) [Designated +RA} or Vehicle (DMSO[Designated —RA] or Transduction of the UBE1L Retrovirus, Designated+UBE1L] as Compared to Transduction of the Same Retrovirus Without anInsert (Designated −UBE1L] Cell line NBT, % NB4-S1 (−RA) 0 NB4-S1 (+RA)92 NB4-S1 (−UBE1L) 0 NB4-Sl (+UBE1L) 0Promotion of apoptosis was not observed in HeLa cells transduced witheither the UBE1L or insertless retroviral vector. Thus, UBE1Ltransduction preferentially triggered apoptosis in PML/RARα-expressingcells. Induction of apoptosis was so rapid, however, that examination ofthe mechanisms signaling apoptosis was precluded.

As demonstrated herein, there is a tight link between UBE1L inductionand PML/RARα degradation. More specifically, experiments describedherein are demonstrative of an antagonistic relationship between UBE1Land PML/RARα. Further, increases in expression of UBE1L rapidly induceapoptosis in cells expressing PML/RARα. Accordingly, UBE1L is believedto be a target for repression by PML/RARα and induction of apoptosis incells expressing this oncogenic protein.

Additional experiments in BEAS-2B human bronchial epithelial cellsconfirmed that co-transfection of UBE1L with transfected cyclin D1triggers degradation of the oncogenic protein cyclin D1. Thus, thedegradation program described herein is active beyond leukemia. In thisstudy β-actin was used as a control to confirm that similar amounts oftotal protein were added per lane. Prior work has implicated cyclin D1degradation as a chemopreventive target (Langenfeld et al. Proc. Natl.Acad. Sci. USA 94: 12070-12074; Boyle et al. J. Natl Cancer Inst. 199991: 373-379). Thus, these experiments directly implicate UBE1L in cancerchemoprevention. The deconjugase UBP43 is also implicated in thedescribed degradation program in that transfection of UBP43 stabilizescyclin D1 despite transfection of UDE1L in BEAS-2B human bronchialepithelial cells.

RA also augments the ubiquitin-like protein ISG15 expression in RAsensitive but not resistant NB4 cells. In addition, RA-treatmentincreases intracellular ISG15 conjugation in retinoid-sensitive NB4cells. This is indicative of a link between increased ISG15 and UBE1Lexpression and induction of myeloid differentiation. Consistent withthis is that RA-treatment increases intracellular ISG15 conjugation inretinoid-sensitive NB4 cells. A physical interaction between UBE1L andISG15 was established in vivo using a transient co-transfection assay.This interaction was not observed when mutant ISG15 lacking essentialc-terminal glycines was examined. Thus, these experiments are indicativeof a coordinate regulation of UBE1L and ISG15. Accordingly, it isbelieved that, like UBE1L, induction of ISG15 can also enhancedegradation of oncogenic proteins such as PML/RARα and promote apoptosisof cancer cells expressing these oncogenic proteins.

Agents that selectively induce UBE1L and/or ISG15 expression and/oractivity in APL are thus expected to cause antileukemic effects bytriggering PML/RARα degradation and apoptosis. Based on findingspresented here and previous reports (Yuan, W. and Krug, R. M. EMBO 200120: 362-371), UBE1L is also expected to have an important biologicalrole beyond APL. UBE1L maps to chromosome 3p, a region frequentlydeleted in lung cancers; UBE1L repression is frequent in lung cancers(Kok et al. Proc. Natl. Acad. Sci. USA 1993 90: 6071-6075; Carritt etal. Cancer Res. 1992 52: 1536-1541) where it may exert a tumorsuppressive effect. This possibility, when coupled with the expressionpattern of UBE1L in human tissues or tumor cells (McLaughlin et al. Int.J. Cancer 2000 85: 871-876) and results of UBE1L retroviral transductionreported herein, indicate that UBE1L regulates growth of both normal andneoplastic or pre-neoplastic cells.

PML/RARα degradation is also inhibited by UBP43 transfection asconfirmed by co-transfection experiments with PML/RARα and UBP43 whereUBP43 was able to overcome the ability of UBE1L to trigger degradationof PML/RARα. Accordingly, agents that selectively inhibit the expressionand/or activity of the deconjugase UBP43 are also expected to triggerPML/RARα degradation and apoptosis in cancer cells expressing PML/RARα.

Cancer cells, and in particular RA sensitive cancer cells or cancercells expressing oncogenic proteins such as PML/RARα can be contactedwith agents of the present invention that induce UBE1L and/or ISG15expression and/or activity or inhibit the expression and/or activity ofthe deconjugase UBP43 to trigger degradation of oncogenic proteins suchas PML/RARα and apoptosis in the cancer cells. Activation of this newlyidentified pathway is also expected to trigger degradation of otheroncogenic proteins in non-leukemia cells, including other neoplastic orpre-neoplastic cells. In a preferred embodiment agents used selectivelyinduce UBE1L and/or ISG15 activity and/or expression or selectivelyinhibit UBP43 activity and/or expression.

Compositions of the present invention comprising an agent which inducesactivity and/or expression of UBE1L or ISG15 or inhibits UBP43expression and/or activity can be administered to a patient to preventor treat cancer, and in particular cancers such as APL which express theoncogenic protein PML/RARα. In a preferred embodiment, the agentselectively induces activity and/or expression of UBE1L or ISG15 orselectively inhibits UBP43 as selectivity of these agents shouldalleviate undesirable clinical toxicities that complicate RA or arsenictrioxide treatments. Compositions of the present invention preferablyfurther comprise a pharmaceutically acceptable vehicle selectedroutinely by those of skill in the art based upon the type of cancerbeing treated and the route of administration best suited for treatmentof that type of cancer. Effective amounts of the agent to beadministered can be determined routinely by those of skill in the artbased upon in vitro and in vivo assays demonstrative of pharmacologicalactivity such as those described herein.

Agents for use in the compositions of the present invention which induceexpression and/or activity of UBE1L and/or ubiquitin-like proteins suchas ISG15 or inhibit the deconjugase UBP43 can be identified routinely bythose of skill in the art in accordance using in vitro assays such asdescribed herein. For example, test agents can be screened for theirability to induce UBE1L expression via reverse transcription-PCR asdescribed herein which measures increases in UBE1L mRNA or immunoblotexpression as described herein which measures UBE1L protein in thepresence and absence of a test agents. An increase in UBE1L mRNA orprotein levels in the presence of the test agent as compared to UBE1LmRNA or protein levels in the absence of the test agents is indicativeof the test agent inducing UBE1L and being potentially useful as ananticancer agent. Similar assays can be performed to identify agentswhich induce ubiquitin-like proteins such as ISG15. Agents which inhibitthe deconjugase UBP43 can also be identified routinely. Such agents canthen be administered to prevent and treat cancer.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Cell Culture and Induction Protocol

RA and dimethyl sulfoxide (DMSO) were purchased from Sigma ChemicalCompany (St. Louis, Mo.). Stock RA (10 mM) solutions were dissolved inDMSO, stored in liquid nitrogen, and used in the dark duringexperiments. RPMI 1640 and D-MEM were purchased from Cellgro/Mediatech(Herndon, Va.). The NB4 APL cell line expresses PML/RARα (Lanotte et al.Blood 1991 77: 1080-1086). NB4-S1 and NB4-R1 are RA-sensitive andRA-resistant clones of NB4 cells, respectively (Nason-Burchanel et al.Differentiation 1997 61: 321-331). These cells were cultured in RPMI1640 media supplemented with 10% FBS as described by Nason-Burchenal etal. (Differentiation 1997 61: 321-331). Chinese hamster ovary (CHO)cells were cultured in D-MEM supplemented with 5% FBS, 100 units/mlpenicillin, 100 units/ml streptomycin, and 2 mM L-glutamine in a 5% CO₂humidified incubator at 37° C. Human bronchial epithelial cells(BEAS-2B) were cultured in serum-free LHC-9 medium (Biofluids,Rockville, Md.) in accordance with established techniques (Langenfeld etal. Proc. Natl. Acad. Sci. USA 94: 12070-12074; Boyle et al. J. NatlCancer Inst. 1999 91: 373-379). HeLa cells were cultured in DMEMsupplemented with 10% FBS, 100 units/ml penicillin, 100 units/mlstreptomycin, and 2 mM L-glutamine in a 5% CO₂, humidified incubator at37° C.

Example 2 Differentiation and Apoptosis Markers

NB4 cell differentiation was scored by using the nitroblue tetrazolium(NBT) reduction assay (Nason-Burchenal et al. Differentiation 1997 61:321-331; Nason-Burchenal et al. Blood 1998 92: 1758-1767;Nason-Burchanel et al. Oncogene 1998 17: 1759-1768) Transductants wereidentified by green fluorescent protein (GFP) coexpression. Apoptosiswas scored by using established techniques and Hoechst staining oftransductants that co-expressed GFP (Nason-Burchenal et al. Blood 199892: 1758-1767; Nason-Burchenal et al. Oncogene 1998 17: 1759-1768;Stadheim et al. Cancer Res. 2001 61: 1533-1540). Digital images werecollected by using an Olympus 1X70 inverted microscope, a cooledcharge-coupled device camera, and a MiraCal Pro Single Cell ImagingSystem (Olympus LSR Research, Melville, N.Y.).

Example 3 Plasmid Constructs

A full length UBE1L cDNA containing plasmid was obtained in accordancewith the method of Kote et al. (Gene Expression 1995 4: 163-175). ThepGEM-HA-1E1 plasmid was obtained in accordance with the method ofHandley et al. (Proc. Natl. Acad. Sci. USA 1991 88: 250-262).pSG5-HA-1E1 was constructed by cloning the HA-1E1 fragmented into thepSG5 expression vector. An EcoR1 fragment containing the UBE1L cDNA wascloned into EcoR1-restricted pSG5 to yield the pSG5-UBE1L plasmid. Atruncated UBE1L plasmid (UBE1L-T) lacks an EclXI/SnaBI fragment in thecarboxy terminus of UBE1L. The hemagglutinin (HA)-tagged PML/RARαexpression vector was constructed from pCMX-PML/RARα and pCMV-HA(CLONTECH) plasmids. The pGL3-UBE1L Luc reporter plasmid contained theluciferase gene and 5′ promoter elements of UBE1L. It was constructed byusing a PCR amplified fragment of the UBE1L promoter derived from NB4-S1genomic DNA (forward primer: 5′-GCAACCGAGTGAGACTGTCT-3′ (SEQ ID NO:1);reverse primer 5′-GCGCTCAGAGATAGGGTTT-3′ (SEQ ID NO:2)). DNA sequenceanalysis confirmed this cloning.

Example 4 UBE1L mRNA Expression Assays

UBE1L mRNA expression was assessed by a reverse transcription-PCR assayin accordance with established methods (Kakizuka et al. Cell 1991 68:663-674). The forward primer was 5′-AGGTGGCCAAGAACTTGGTT-3′ (SEQ IDNO:3), and the reverse primer was 5′CACCACCTGGAAGTCCAACA-3′ (SEQ IDNO:4). The PCR product was visualized by probing with a ³²P-labeledprimer. Results were confirmed independently by Northern analysis usinga 1.0-kb EcoR1/NcoI-radiolabeled UBE1L probe in accordance with standardtechniques (Langenfeld et al. Proc. Natl. Acad. Sci. USA 1997 94:12070-12074). This probe had limited homology to El.

Example 5 Generation of Anti-UBE1L Antisera

Two rabbit polyclonal antibodies against UBE1L were independentlyderived (Covance Research Products, Denver, Pa.) using one peptidewithin the amino terminus (DCDPRSIHVREDGSLEIGD (SEQ ID NO:5)) and asecond peptide within the carboxyl terminus (PGSQDWTALRELLKLL (SEQ IDNO:6)). Specificities of these antisera were confirmed by immunoblotanalyses of UBE1L-transfected CHO cells.

Example 6 Immunoblot Analysis

Immunoblot analyses were performed using established techniques(Langenfeld et al. Proc. Natl. Acad. Sci. USA 1997 94: 12070-12074;Spinella et al. J. Biol. Chem. 1999 274: 22013-22018). Anti-RARαantibody was provided by and can be purchased from P. Chambon (InstitutNational de la Santé et de la Recherche Médicale, Strasbourg, France) todetect PML/RARα (Nason-Burchenal et al. Blood 1998 92: 1758-1767;Nason-Burchenal et al. Oncogene 1998 17: 1759-1768). An anti-HA mAb waspurchased (Babco, Richmond, Calif.) as was an anti-actin polyclonalantibody, C-11 (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Example 7 Transfection Procedure

Transient transfection of BEAS-2B or CHO cells was accomplished by usingEffectene and transfection methods in accordance with the manufacturer'sinstructions (Qiagen, Valencia, Calif.). A β-galactosidase reporterplasmid (pCH111) was cotransfected to control for transfectionefficiencies.

Example 8 Retroviral Constructs and Transduction Procedures

MSCV-IRES-GFP was constructed to express UBE1L cDNA by cloning an EcoR1fragment from pSG5-UBE1L into an EcoR1 site of this retroviral vector.Restriction endonuclease and partial DNA sequence analyses confirmedcloning was in the desired orientation. A vector without an insertserved as a control. For each vector, 10 μg was transiently transfectedusing calcium phosphate precipitation along with the CellPhectTransfection kit (Amersham Pharmacia). The 293GPG packaging cell linewas provided by R. Mulligan (Harvard University, Cambridge Mass.) and isavailable to other investigators. Forty-eight hours later viralsupernatant from 293GPG transfectants (Ory et al. Proc. Natl. Acad. Sci.USA 93: 11400-11406) was used to transduce NB4-S1 or HeLa cells in thepresence of 6 μg/ml polybrene (Sigma Chemical Company, St. Louis, Mo.).Twenty-four hours later, FACS analysis was performed, and cells positivefor GFP expression were harvested by sorting and used for theseexperiments.

1. A composition for prevention or treatment of cancer comprising anagent which induces UBE1L or a ubiquitin-like protein ISG15 or an agentwhich inhibits the deconjugase UBP43.
 2. The composition of claim 1wherein the agent selectively induces UBE1L or a ubiquitin-like proteinISG15 or selectively inhibits the deconjugase UBP43.
 3. A method forenhancing pro-apoptotic and degradative pathways of neoplastic orpre-neoplastic cells comprising contacting cells with the composition ofclaim
 1. 4. A method for enhancing pro-apoptotic and degradativepathways-of-neoplastic or pre-neoplastic cells comprising contactingcells with the composition of claim
 2. 5. A method for preventing ortreating cancer in a patient comprising administering to a patient thecomposition of claim
 1. 6. A method for preventing or treating cancer ina patient comprising administering to a patient the composition of claim2.
 7. A method for identifying agents for use in preventing or treatingcancer comprising determining an agent's ability to induce UBE1L or aubiquitin-like protein ISG15 or to inhibit a deconjugase UBP43.